CN103223176B

CN103223176B - miR-24 and application of inhibitor of miR-24

Info

Publication number: CN103223176B
Application number: CN201310105830.6A
Authority: CN
Inventors: 金由辛; 秦文明
Original assignee: Shanghai Institutes for Biological Sciences SIBS of CAS
Current assignee: Suzhou Zhongke Cell Transformation Research Institute
Priority date: 2009-05-26
Filing date: 2009-05-26
Publication date: 2015-03-25
Anticipated expiration: 2029-05-26
Also published as: CN103223176A

Abstract

The invention provides a method for adjusting and controlling FAF1 genetic expressions. Cancer cells in cancers such as a prostate cancer can be induced to be dead through adjustment and control of the FAF1gene expressions. The method comprises the steps of providing miR-24 inhibitor, and/or simultaneously mutating a binding site of miR-24 in FAF1 genes. The method further relates to a drug composition containing the miR-24 inhibitor and a kit.

Description

The application of miR-24 and inhibitor thereof

The application be applicant in the application number that on May 26th, 2009 submits to be 200910051968.6, denomination of invention is the divisional application of the application for a patent for invention of " method and composition of regulation and control FAF1 gene and the purposes of described compositions ".

Technical field

The present invention relates to miRNA field.Specifically, the present invention relates to the method and composition of regulation and control FAF1 gene and the purposes of described compositions.

Background technology

MicroRNA is the micromolecule non-coding RNA of the endogenous expression of a class, about about 22nt, and they are regulated and controled target gene by post-transcriptional level.Usually they are by shearing mRNA or suppressing the translation of mRNA to regulate and control target gene.Although have been found that some miRNA work in a lot of biological processes, such as, control to grow, embryo generates, cell differentiation propagation and apoptosis, at present the target gene of most miRNA and functional study are not also perfectly clear.

Apoptosis is a kind of situation of the apoptosis in multicellular organisms.Apoptosis embodies the death for the change of a series of biochemical indicator in cell, the metamorphosis of cell and cell.Increasing evidence shows that miRNA can the apoptosis of regulating cell in growth course and in some other biological processes.Have also discovered the miRNA that some are relevant to apoptosis simultaneously.But the miRNA of all known modulating apoptosis is the 3 ' untranslated region by being attached to target gene to be carried out regulating and controlling.Also be not combined in the apoptotic example of regional control beyond target gene mRNA3 ' untranslated region at present.

FAF1 gene is a composition of apoptosis-inducing signal complex (DISC), although it does not have typical apoptosis domain, it can regulate and control apoptosis with caspase8 and FADD protein binding.Nearest research finds depositing in case without any exogenous apoptosis inducing factor, and in Jurkat cell, in L cell and BOSC23 cell, high expressed FAF1 gene all can cause the apoptosis of cell.Existing document is reported, and stomach organization cell and cervical cancer tissues FAF1 express and decline, and decline relevant with the low differentiation of cancerous cell and level of apoptosis; The expression in the prefrontal cortex (frontal cortex) of parkinson disease (PD) patient and Alzheimer (AD) patient of the expression of FAF1 obviously raises relative to matched group.Existing bibliographical information, " positive expression rate (37.50%) of the FAF1 of stomach organization is starkly lower than cancer beside organism (62.50%, P < 0.05) and normal structure (72.50%, P < 0.01); Low or do not break up stomach organization FAF1 positive expression rate significantly lower than senior middle school differentiation stomach organization (73.08%vs64.29%, P < 0.05).Apoptosis in gastric cancer index relevant with Carcinoma cell differentiation degree, metastasis, intramural invasion and clinical stages (the equal < 0.05 of P).FAF1 expresses the apoptosis in gastric cancer index of negative group significantly lower than the positive group (P < 0.01) of FAF1.Conclusion: stomach organization cell FAF1 expresses decline, and declines relevant with the low differentiation of cancerous cell and level of apoptosis, prompting FAF1 low expression may occur with gastric cancer and develop relevant, affects cell differentiation and apoptosis is its possible mechanism of action." in addition, known miR-24 exists high expressed in colon cancer and cancer of pancreas.

Carcinoma of prostate (PCa) is the kinds of tumor of male genitourinary system, and its sickness rate ranks first American-European countries is high, at the sickness rate of China also in remarkable ascendant trend.In early days, hormone-independent prostate cancer (HDPC) can reach the object reducing tumor body, reduce blood PSA by endocrine therapy, but in treatment after 1 ~ 2 year, be evolved into hormone-independent prostate cancer (HIPC) gradually, cause patient finally to die from the extensive transfer of the insensitive cell of hormone.The treatment of hormone-independent prostate cancer is more difficult, and the meta Overall survival of chemotherapy is only 15.16 ~ 26.16 months.Many patients with prostate cancer, especially hormone-independent prostate cancer patient, start to seek alternative medicine.There is no special effective treatment for hormone-independent prostate cancer at present, herb therapy is one of the most frequently used alternative medicine.Cyto-dynamics research confirms, carcinoma of prostate morbidity is unbalance due to Cell apoptosis and proliferation and cell number increase that is that cause.Many scholars make great efforts to seek crucial apoptosis regulator, to becoming the effective ways for the treatment of of advanced carcinoma of prostate.In addition, the molecular pathway improving apoptosis also has remarkable effect to the vascularization of minimizing carcinoma of prostate, the sensitivity of enhancing tumor to other treatment (as radiotherapy, chemotherapy etc.).

In recent years, along with people are to the further investigation of apoptosis molecular mechanism, recognize that increasing apoptosis of tumor cells is an approach of effectively treating tumor, many antineoplastic chemotherapy medicines destroy tumor cell by cytotoxicity, but can cross resistance be produced between chemotherapeutic and reduce chemotherapy effect, and increasing toxic action.The molecular mechanism and seeking exploring independent prostate cancer cells apoptosis can cause the molecular target of independent prostate cancer cells apoptosis to be the focus of current hormone-independent prostate cancer correlational study.

Summary of the invention

Carcinoma of prostate DU-145 is the cell strain of androgen independent, the application finds the expression being suppressed miR-24 by miR-24-ASO, obviously can cause the apoptosis of DU-145 cell, and the toleration of DU-145 cell to apoptosis-induced medicine star born of the same parents rhzomorph is obviously weakened.Meanwhile, in transfection miR-24-ASO suppress the expression of miR-24 simultaneously cotransfection pcDNA3.1-FAF1 overexpression Faf1 gene can significantly induce the apoptosis of DU-145 cell after 48 hours and apoptosis rate reaches about 70%.Meanwhile, we in HeLa cell, have also discovered the expression suppressing miR-24 can the apoptosis of inducing cell.Up to the present, the miRNA that can regulate and control FAF1 expression is not also found.In this application, we have found miR-24 to be regulated and controled FAF1 gene by the open reading frame region being attached to FAF1mRNA.Meanwhile, miR-24-ASO is used to suppress the expression of miR-24 can cause the apoptosis of DU-145 cell and HeLa cell.The results show, miR-24 can as the Effective target site for the treatment of hormone-independent prostate cancer.MiR-24-ASO can as exploitation treatment the hormone-independent prostate cancer even active drug of some other cancer simultaneously.

In addition, the FAF1 gene (i.e. FAF1-M12) having done samesense mutation at the seed region of miR-24 binding site also can cause a large amount of apoptosis of DU-145 cell (pcDNA3.1-FAF1-M12 in the every hole of transfection 100ng can induce about 34% after 36 hours in 12 orifice plates).Prove to be used alone the apoptosis that FAF1-M12 also can induce prostate gland cancer cell DU-145.

Therefore, one aspect of the present invention relates to a kind of FAF1 gene of separation, the nucleotide sequence of aminoacid sequence shown in this FAF1 gene code SEQ ID NO:2, wherein, correspond in this nucleotide sequence in the base of SEQ ID NO:1 1650-1670 position and/or 2128-2148 bit base to have compared with the described 1650-1670 position of one or more base and SEQ ID NO:1 and/or 2128-2148 bit base and there occurs samesense mutation.

In a preferred embodiment, the nucleotide sequence of the FAF1 gene that the present invention is separated is as shown in SEQ ID NO:1 454-2406 position, but be as the criterion with the sequence numbering of SEQ ID NO:1, the one or more nucleotide in 1650-1670 position wherein and/or 2128-2148 position there occurs samesense mutation.

In another preferred embodiment, the nucleotide sequence of described FAF1 gene is as shown in the 454-2406 position of SEQ ID NO:12.

The present invention relates to the purposes of FAF1 gene in the medicine preparing treatment or prophylaxis of cancer of FAF1 gene or the present invention's separation do not suddenlyd change on the other hand.In a preferred embodiment, described cancer is the cancer of FAF1 gene unconventionality expression.

In a preferred embodiment, the cancer of described FAF1 gene unconventionality expression is selected from malignant mesothe, hormone-independent prostate cancer, cervical cancer, gastric cancer, colon cancer and cancer of pancreas.

Further aspect of the present invention relates to the purposes of miR-24 inhibitor in the compositions preparing treatment or prophylaxis of cancer or test kit.In a preferred embodiment, described cancer is the cancer of FAF1 gene unconventionality expression.

In a preferred embodiment, described miR-24 inhibitor is the antisensenucleic acids of miR-24.In another preferred embodiment, described antisensenucleic acids is as shown in SEQ ID NO:4.

In another preferred embodiment, the cancer of described FAF1 gene unconventionality expression is selected from malignant mesothe, hormone-independent prostate cancer, cervical cancer, gastric cancer, colon cancer and cancer of pancreas.

Another aspect of the invention relates to a kind of expression vector, and this expression vector contains the FAF1 gene that the present invention is separated.

The present invention also relates to the method for the expression of FAF1 gene in a kind of external regulating cell on the other hand, and the method comprises:

(1) suppress the expression of miR-24 in this cell, and/or (2) implement samesense mutation to the miR-24 binding site in FAF1 gene; And/or (3) apply the FAF1 gene of FAF1 gene or the separation according to claim 1 do not suddenlyd change or expression vector according to claim 9; And/or (4) apply the expression product of FAF1 gene; Or (5) overexpression miR-24.

Further aspect of the present invention also relates to the antisensenucleic acids of miR-24.In a preferred embodiment, described antisensenucleic acids is as shown in SEQ ID NO:4.In another preferred embodiment, described anti sense nucleotide sequence is 5 '-ctGTTCCTgctgAACTGAgcCA-3 ', and wherein lower case represents the position of lock nucleic acid marking.

The invention still further relates to the purposes of miR-24 in the compositions of screening treatment or prophylaxis of cancer or test kit.In a preferred embodiment, described cancer is the cancer of FAF1 gene unconventionality expression.

Herein, " cancer of FAF1 gene unconventionality expression " refers to the cancer of the expression of its FAF1 gene higher or lower than the expression of the FAF1 gene in normal cell.Those skilled in the art are easy to judge which kind of is expressed as unconventionality expression according to state of the art.Cancer herein also comprises " cancer of FAF1 gene mediated ", namely cancer generation, form and/or deposit the cancer that FAF1 gene during the course take part in.

Shown in the coding SEQ ID NO:2 that the present invention also comprises separation, the nucleotides sequence of aminoacid sequence is listed in the purposes in the medicine of preparation or screening treatment or prophylaxis of cancer.In a preferred embodiment, described cancer is the cancer of FAF1 gene unconventionality expression.In a preferred embodiment, shown in the coding SEQ ID NO:2 of described separation, the nucleotide sequence of aminoacid sequence is as shown in the 454-2406 position of SEQ ID NO:1.

Other side of the present invention will hereafter be described later in detail.It is pointed out that the combination in any of above-mentioned and each specific features is hereafter also within the scope of the invention.

Accompanying drawing explanation

Fig. 1 shows downward miR-24 can be apoptosis-induced, and affect the propagation of DU-145 cell.A (), with 20nMmiR-24-ASO or NC-ASO process DU-145 cell, with annexin V and iodate third ingot (propidiumiodine) dyeing after 48 hours, measures apoptosis (n=3 ± SE by FACS; * p<0.01).Lower the apoptosis of miR-24 induction of DU-145 cell.B () uses 1 μM of star born of the same parents rhzomorph apoptosis-induced 2 hours (n=3 ± SE with 20nM miR-24-ASO or NC-ASO transfection after 48 hours; * p<0.01).After transfection miR-24-ASO, sensitivity is higher.C (), with after 20nMmiR-24-ASO or NC-ASO transfection, detects cell proliferation (n=3 ± SE) at different time points CCK-8 test kit (Dojindo).Lower the propagation that miR-24 have impact on DU-145 cell.D () cell uses the concentration process of 20nM after 20 hours as mentioned above, carry out western engram analysis caspase8 activity to the mature form of pro-caspase8 and caspase8.After transfection miR-24-ASO, caspase8 is activated.Each experiment in triplicate.

Fig. 2 shows miR-24 and regulates FAF1 by targeting ORF region.A () uses RNA22 software (IBM), the CDS region of prediction Faf1mRNA exists the miRNA miR-24 binding site of two presumptions.Shown in be numbered the numbering of the coded sequence of FAF1 albumen.Show the sequence of people miR-24, binding site and in conjunction with energy.B (), with the report carrier transfection 293T cell be made up of Luciferase luciferase, contains the pGL3 report carrier of miR-24 binding sequence in 3 ' untranslated region of luciferase in 293T transit cell dye.Simultaneously cotransfection CMV-Renilla luciferase carrier, as internal standard.Compared with NC-mimics, miR-24-mimics reduces the expression (n=3 ± SE of the Luciferase luciferase of the miR-24 binding sequence containing prediction; * p<0.01).FAF1 is regulated in (c) miR-24 body.Process 293T cell as shown in FIG., in miR-24-mimics body, reduce the expression of Faf1.The expression of Faf1 albumen has been raised after transfection miR-24-ASO.MiR-24 dose-dependent inhibition Faf1 in (d) display 293T cell.With 100ng pcDNA3.1-FAF1 and 1,5,20nM miR-24-mimics cotransfection 293T cell 24 hours, and immunoblotting as mentioned above.E Faf1 that the miR-24-mimics transfection of () display 1nM or 5nM suddenlyd change after 24 hours fails to be lowered.Although can lower Faf1 a little with 20nM miR-24-mimics transfection, this may be because the mutational site deficiency of Faf1.Each experiment repetition 3 times.

Fig. 3 shows, and miR-24 regulates apoptosis by targeting FAF.As shown in the figure with different agent treated DU-145 cells, reagent transfection measured apoptosis (n=3 ± SE, * p<0.01) through FACS after 48 hours.Lower miR-24 not only induction of the apoptosis of DU-145, process LAN Faf1 is also induction of the apoptosis of DU-145.As cotransfection pcDNA3.1-FAF1 and miR-24-ASO, the ratio of apoptotic cell increases greatly.The apoptosis that process LAN FAF1 causes is saved by miR-24-mimics.Above-mentioned data display, miR-24 process LAN can protect DU-145 cell that the apoptosis of FAF1 induction does not occur, and lowers the apoptosis that miR-24 can increase the DU-145 cell of FAF1 induction.(for 12 orifice plates, the concentration of carrier transfection is the concentration of every hole 100ng, mimics and ASO transfection is every hole 20nM).

Fig. 4 shows miR-24 and regulates apoptosis by the ORF region in conjunction with FAF1 gene.As shown in FIG. with different agent treated DU-145 cells.Apoptosis (n=3 ± SE, * p<0.01, * * p<0.05) is measured with FACS after 36 hours with reagent transfection.Being used in the FAF1 gene (i.e. pCDNA3.1-FAF1-M12) that miR-24 binding site done samesense mutation does not have the FAF1 gene suddenlyd change that DU145 cell can be induced to occur more apoptosis than being used alone.The apoptosis that the FAF1 that process LAN miR-24 can not protect DU-145 Leukopenia to express sudden change induces.(for 12 orifice plates, the concentration of carrier transfection is the concentration of every hole 100ng, mimics and ASO transfection is every hole 20nM).

Fig. 5 shows, and lowering miR-24 also affects the propagation of HeLa cell and the sensitivity to star born of the same parents rhzomorph.A () uses 20nM miR-24-ASO or NC-ASO transfection after 48 hours, with 1 μM of star born of the same parents rhzomorph cell death inducing, 2 hours (n=3 ± SE; * p<0.01).After transfection miR-24-ASO, sensitivity becomes higher.At different time CCK-8 kit detection cell propagation (n=3 ± SE) after (b) display transfection 20nMmiR-24-ASO or NC-ASO.

Fig. 6 shows use Interfer IN transfection reagent respectively to transfection 20nM miR-24-ASO in two strain stomach cancer cell HGC-27 and MGC-803, the result that after 36 hours, basis of microscopic observation arrives.

Fig. 7 shows the Annexin V-FITC and PI coloration result and apoptosis rate that test described in Fig. 6.

In Fig. 1 a, Fig. 1 b, Fig. 3 and Fig. 4, Fig. 5, Fig. 7, the abscissa of facs analysis figure, vertical coordinate and scale are all identical, and abscissa is annexin V-FITC, and vertical coordinate is PI, and scale is 10 ⁰-10 ⁴.Western blot in all figure tests all using GAPDH as internal reference.

Detailed description of the invention

FAF1 gene is also called FAS binding factor 1, and be a composition of apoptosis-inducing signal complex (DISC), its nucleotide sequence is as SEQ ID NO:1(Genebank accession number NM_007051) shown in, aminoacid sequence is as shown in SEQ IDNO:2.454-2406 position in SEQ ID NO:1 is the open reading frame region of encoding proteins.FAF1 gene has expression in most mammal is as the normal structure in the animal such as people and large mice, be included in the various cells of the various tissue of mammal, but expression can reduce in the tumor cells such as malignant mesothe.

The FAF1 gene of a kind of separation of first aspect present invention, the nucleotide sequence of aminoacid sequence shown in its coding SEQ ID NO:2, wherein, correspond in this nucleotide sequence in the base of SEQ ID NO:1 1650-1670 position and/or 2128-2148 bit base to have compared with the described 1650-1670 position of one or more base and SEQ ID NO:1 and/or 2128-2148 bit base and there occurs samesense mutation.Samesense mutation can be identical, also can be different: the position residing for the base of namely undergoing mutation can be identical, also can be different; And/or the quantity of the base of undergoing mutation can be identical, also can be different.Preferably, the base quantity of undergoing mutation in each binding site is that 1-21 is individual, such as, be that 2-18 is individual, 2-15 is individual, 2-12 is individual, 2-10 is individual, 2-8 is individual, 2-7 is individual, 2-5 is individual.Preferably, described sudden change occurs in the 2 to the 8 (from 3 ' to 5 ') in two corresponding regions, and 1 namely in 2-8 position or several base there occurs sudden change.

Samesense mutation refers to annex phenomenon because the genetic codon of biology exists, and after a certain sequence change, is translated into same aminoacid, is namely called samesense mutation in certain original amino acid whose position.When relating to a peptide species, " separation " means that described molecule is separated and separates from the naturally occurring whole organism of this molecule of discovery, or substantially there is not the biomacromolecule of other identical type.Term " separation " for nucleic acid is: a kind of nucleic acid molecules, and it lacks the sequence of combination natural with it wholly or in part; Or a sequence, because its natural existence, but there is the heterologous sequence with its combination; Or from the molecule of chromosome separation.

More specifically, the nucleotide sequence of the FAF1 gene of separation of the present invention is as shown in SEQ ID NO:1 454-2406 position, but the one or more nucleotide in 1650-1670 position wherein and/or 2128-2148 position there occurs samesense mutation.Described nucleotides number is all be as the criterion with the numbering in SEQ ID NO:1.

The samesense mutation occurred in described 1650-1670 position and 2128-2148 position can be identical, and also can be different: the position residing for the base of namely undergoing mutation can be identical, also can be different; And/or the quantity of the base of undergoing mutation can be identical, also can be different.Preferably, the base quantity that samesense mutation occurs in each binding site is that 1-21 is individual, such as, be that 2-18 is individual, 2-15 is individual, 2-12 is individual, 2-10 is individual, 2-8 is individual, 2-7 is individual, 2-5 is individual.Preferably, described samesense mutation occurs in the 2 to the 8 (from 3 ' to 5 ') in two basic change site areas, and 1 namely in 2-8 position or several base there occurs samesense mutation.In FAF1 gene of the present invention, can only in two basic change site any one occur samesense mutation, also all can there is samesense mutation in two basic change site.

Except occurring except samesense mutation in above-mentioned 1650-1670 position and/or 2128-2148 position, the nucleotide of other position of FAF1 gene of the present invention, such as, one or more nucleotide in 454-1649 position, 1671-2127 position and 2149-2406 position nucleotide also can be undergone mutation, as long as this sudden change does not have influence on gained FAF1 gene function had in the present invention.Specifically, the present invention also comprises the FAF1 gene of coding following proteins: in the aminoacid sequence of described 454-1649 position, 1671-2127 position and 2149-2406 position nucleotide coding through replacement, lack or add one or several aminoacid and still remain with the activity of albumen shown in SEQ ID NO:2 by protein protein derived shown in SEQ ID NO:2.

In a preferred embodiment, FAF1 gene of the present invention is as shown in the 454-2406 position in SEQ ID NO:12.

Various method can be adopted to prepare protein of the present invention.Usually protein of the present invention is prepared with recombination method.The polynucleotide of coding present protein can be prepared by standard molecular biology method.Such as, the polynucleotide sequence of above-mentioned molecule of encoding can be obtained with recombination method, such as, pass through cell screening cDNA and genomic library from expressing this gene, or by deriving this gene from the known carrier comprising this gene.Also can synthesize but not clone the interested gene of preparation.The codon of available suitable particular sequence digests this molecule.Then the oligonucleotide of the overlap prepared with standard method is assembled complete sequence, and be fitted in complete coded sequence.See such as Edge (1981) Nature292:756; Nambair etc. (1984) Science223:1299; With (1984) J.Biol.Chem.259:6311 such as Jay.

Therefore, concrete nucleotide sequence can be obtained from the carrier carrying required sequence, or with various oligonucleotide synthesis method known in the art, as site-directed mutagenesis and polymerase chain reaction (PCR), synthesize wholly or in part.See such as Sambrook, " molecular cloning: laboratory manual " (Molecular Cloning:a Laboratory Manual), the second edition, 1989.Specifically, the method obtaining the nucleotide sequence of the required sequence of coding is the oligonucleotide of the synthesis of the overlap of the annealing complementary sets prepared with the automatic polynucleotide synthesizer of routine, then connect with suitable DNA ligase, and to increase the nucleotide sequence connected with PVR.See such as (1991) Proc.Natl.Acad.Sci.USA88:4084-4088 such as Jayaraman.In addition, also synthesis (the Jones etc. of oligonucleotide orientation can be used in the present invention, (1986) Nature54:75-82), first have the oligonucleotide site directed mutagenesis of nucleotide region (Riechmann etc., (1988) Science239:1534-1536 such as (1988) Nature332:323-327 and Verhoeyen) and with the enzymatic that T4DNA polymerase carries out fill notched oligonucleotide synthesis (Queen etc. (1989) Proc.Natl.Acad.Sci.USA86:10029-10033).

Once prepare or be separated coded sequence, just these sequence clones can be entered in any suitable carrier or replicon.To those skilled in the art, various cloning vehicle is known, and the screening of suitable cloning vehicle is select permeability.Suitable carrier comprises (but being not limited to): plasmid, phage, transposon, cosmid, chromosome or when with suitable control element in conjunction with time reproducible virus.

Then, under cloned sequence being placed in the control of suitable control element, this depends on the system for expressing.Therefore, under coded sequence can being placed in the control of promoter, ribosome binding site (for bacterial expression) and optionally operon, thus by suitable transformant, interested DNA sequence is transcribed in RNA.This coded sequence can comprise or not comprise signal peptide or targeting sequencing (can process removing upon translation by host subsequently).See such as U.S. Patent No. 4,431,739; 4,425,437; 4,338,397.

Except control sequence, adjustment sequence can be added, thus can relative to the expression of the growth regulating sequence of host cell.Regulate sequence to be well known by persons skilled in the art, the example comprises those and response chemistry or physical stimulation can be caused (comprising the existence regulating compound) to start or close the adjustment sequence of gene expression.The regulating element of other type also can be there is in carrier.Such as, enhancer element can be used to increase the expression of construction.Example comprises SV40 early gene enhancer (Dijkema etc. (1985) EMBO J.4:761); From the enhancers/promoters (Gorman etc. (1982) Proc.Natl.Acad.Sci.USA79:6777) that the sarcoma viral long terminal repeat of Rous (LTR) is derivative; With the element (Boshart etc., (1985) Cell41:521) derived from people CMV, as the element (U.S. Patent No. 5,688,688) that CMV intron A sequence comprises.The origin of replication, one or more selectable labels, one or more restriction site, the potential energy of high copy number amount and the strong promoter that independently copy in suitable host cell also can be included in expression cassette.

Construction of expression vector, make concrete coded sequence be arranged in this and there is the carrier being applicable to regulating sequence, the position of the coded sequence relevant with control sequence and orientation make coded sequence transcribe under " control " of control sequence (that is, this coded sequence of rna polymerase transcribe being incorporated into DNA molecular in control sequence).May need to carry out modification to realize this object to the sequence of interest encodes molecule.Such as, may need in some cases to modify this sequence, thus make it be connected to the control sequence of applicable orientation, namely maintain frame.Before being inserted into carrier, control sequence and other adjustment sequence may be connected to coded sequence.Or, coded sequence Direct Cloning can be entered to comprise in the expression vector of control sequence and suitable restriction site.

By the one or more nucleotide lacking the sequence of the polypeptide of a part of interest encodes, insertion sequence and/or substitute in this sequence, can for the preparation of the mutant of the albumen of the present invention analyzed or analog.The method of modified nucleotide sequence, if site directed mutagenesis etc. is well-known to those skilled in the art.See such as Sambrook etc., above-mentioned; Kunkel, T.A. (1985) Proc.Natl.Acad.Sci.USA (1985) 82:448; Geisselsoder etc. (1987) BioiTechniques5:786; Zoller and Smith (1983) MethodsEnzymol.100:468; Dalbie-McFarland etc. (1982) Proc.Natl.Acad.Sci USA79:6409.

This molecule can be expressed in a variety of systems, comprises insecticide well known in the art, mammal, antibacterial, virus and yeast expression system.Such as, insect cell expression system is well known by persons skilled in the art as rhabdovirus system, is described in as Summers and Smith, Texas Aguricultural Experiment StationBulletin No.1555 (1987).Materials and methods for baculovirus/insect cell expression system all can be buied in a kit form, especially Invitrogen, San Diego CA (" MaxBac " test kit).Similarly, antibacterial and mammalian cell expression system are also well known in the art, and its description is shown in as Sambrook etc., as above.Yeast expression system is also well known in the art, and it describes sees as " Yeast Genetics engineering " (Yeast GeneticEngineering) Butterworths, the London such as (Barr editor, 1989).

Many host cells be applicable to for said system are also known.Such as, mammal cell line is known in the art, it comprises the cell line of the immortalization that can obtain from American type culture collection (ATCC), as (but being not limited to) Chinese hamster ovary cell (CHO), Hela cell, young hamster kidney (BHK) cell, monkey-kidney cells (COS), HEKC, human liver cell cancerous cell (as Hep G2), Madin-Darby Ren Bovis seu Bubali (" MDBK ") cell etc.Similarly, in expression constructs of the present invention, also bacterial host can be used, as escherichia coli, bacillus subtilis and streptococcus.Also available yeast host in the present invention, particularly including saccharomyces cerevisiae (Saccharomycescerevisiae), Candida maltosa (Candida maltosa), Candida albicans (Candida albicans), multiple-shaped nuohan inferior yeast (Hansenula polymorpha), Kluyveromyces fragilis (Kluyveromyces fragilis), Kluyveromyces lactis (Kluyveromyces lactis), season is Meng Shi Pichia sp. (Pichiaguillerimondii) also, pichia pastoris phaff (Pichia pastoris), foxtail millet wine pombe (Schizosaccharomyces pombe) and yarrowia lipolytica.The insect cell that can use together with baculovirus expression system is particularly including Aedes aegypti (Aedes aegypti), Autographa californica multicapsid nucleopolyhedrosisvirus (Autographacalifornica), silkworm (Bombyx mori), black-tailed fruit flies (Drosophila melanogaster), fall army worm (Spodoptera frugiperda) and cabbage looper (Trichoplusia ni).

By the method for various gene delivery well known in the art, the nucleic acid molecules comprising interested nucleotide sequence stably can be integrated on the stable additive type element in host cell gene group or in suitable host cell and maintain.See such as U.S. Patent No. 5,399,346.

According to selected expression system and host, under the condition of marking protein, cultivate and prepare this molecule with the host cell that expression vector as above transforms.Then the protein of expression is isolated and purification from host cell.If expression system by protein secreting in culture medium, then direct from culture medium purified product.If not what secrete, be then separated from cell pyrolysis liquid.The condition of culture be applicable to and the selection of recovery method are all within those skilled in the art's ability.

Therefore, the present invention also comprises the expression vector containing FAF1 gene of the present invention.

Second aspect present invention relates to the method for regulation and control FAF1 gene expression, and the method comprises the expression of the miR-24 of controlled plant.

MiR-24 is a microRNA very large with human tumor dependency, and its nucleotides sequence is classified as 5 '-UGGCUCAGUUCAGCAGGAACAG-3 ' (SEQ ID NO:3).Prior art finds, and miR-24 can not only suppress the expression of tumor suppressor gene p16, and it also has unconventionality expression in some tumor types.Our research finds that miR-24 can be regulated and controled FAF1 gene by two basic change site on the ORF frame that is attached to FAF1.These two sites are respectively 1650-1670 position and 2128-2148 position in present position in SEQ IDNO:1.Usually, a miR-24 is in conjunction with a site.When by some site mutations of FAF1 gene, single miR-24 also can be incorporated on another non-mutational site of the FAF1 gene of this sudden change, and the expression of the FAF1 gene of this sudden change is still subject to the regulation and control of miR-24.In this case, with compared with the FAF1 gene suddenlyd change, the FAF1 genes amplification that the apoptotic ability of the gene induced DU-145 of FAF1 that overexpression suddenlys change is not suddenlyd change relative to overexpression.

In this article, object comprises mammal, its tissue or cell.Mammal comprises people, rat, mice etc." adjustment " comprises mediation and lowers.Adjustment can be regulate or external adjustment in body.When regulating in body, can by compositions of the present invention by the various mode known, such as injection etc., give object.During external adjustment, the present composition and object such as in vitro tissue or cell that contain miR-24 inhibitor can be cultivated altogether, make the active component in compositions enter in vitro tissue or cell; Or adopt known technology active component to be transfected into object as in vitro tissue or cell.Regulate and also comprise overexpression miR-24.

In the present invention, the active component of compositions comprises agonist and the inhibitor of miR-24.Agonist can strengthen the expression of miR-24, thus can suppress the expression of FAF1 better, and result inhibits the apoptosis of cell.In another embodiment, by giving miR-24 inhibitor to realize the adjustment to FAF1.Inhibitor can suppress the expression of miR-24, increases the expression of FAF1, causes FAF1 expressing quantity to increase, thus promotes the apoptosis of cell.The inhibitor of the agonist of the expression of any miR-24 of enhancing and the expression of any miR-24 of suppression all can be used for the present invention.

Third aspect present invention relates to the apoptotic method of adjustment, and the method comprises the expression regulating FAF1 gene.

In this respect, regulate also to comprise being in harmonious proportion and lower, namely for the cell of certain total amount, reduce apoptosis quantity or ratio, or increase apoptosis quantity or ratio.Regulate the method for FAF1 gene expression to comprise (1) and samesense mutation is implemented, to stop miR-24 to the suppression of FAF1 gene to two miR-24 binding sites on FAF1 gene ORF frame; And/or (2) apply agonist or the inhibitor of miR-24; (3) apply FAF1 gene of the present invention or the carrier containing this gene, or apply FAF1 gene expression product; And/or (4) overexpression miR-24.

Cell can be the cell of mammal such as people, mice, rat etc.By regulating the apoptosis of cell, tumor can be treated.Or, by regulating the apoptosis of cell, various animal model for tumour can be built, for developing new drug etc.

Apply the compositions that the agonist of miR-24 or the method for inhibitor comprise agonist or the inhibitor provided containing miR-24, and by said composition body or externally give cell; Or body is interior or in-vitro transfection enters cell.

Method foreign DNA being transfected into mammalian cell is that this area is known, is broadly divided into two large classes, i.e. biological method and physico-chemical process.Conventional in physico-chemical process have calcium phosphate method, microinjection, electroporation, liposome-mediated transfection, and what occur recently take high molecular polymer as the gene delivery techniques of carrier.Biological ways mainly using virus as carrier, by the mode of viral infection by Exogenous DNA transfered in cell, wherein with retrovirus and Adenovirus Transfection system the most conventional.

Calcium phosphate, under good transfection conditions, can reach higher transfection efficiency in the isocellular transfection of 293T.In addition, transfection method also comprises cationic-liposome method, and the liposome and the electronegative phosphate group of nucleic acid that are specially positively charged form complex by cell endocytic.The cationic-liposome being applicable to cationic-liposome method is that this area is known, and can buy from various commercial sources.

Fourth aspect present invention comprises the method for Therapeutic cancer, and the method comprises the expression regulating FAF1, impels cancer cell apoptosis, thus Therapeutic cancer.Cancer comprises the various apoptotic cancer had via FAF1 mediation, includes but not limited to malignant mesothe, hormone-independent prostate cancer, cervical cancer, gastric cancer, colon cancer and cancer of pancreas etc.Regulate the method for the expression of FAF1 to comprise (1) and samesense mutation is implemented, to stop miR-24 to the suppression of FAF1 gene expression to two miR-24 binding sites on FAF1 gene ORF frame; And/or (2) apply miR-24 inhibitor; And/or apply FAF1 gene of the present invention or containing the carrier of this gene, or apply the expression product of FAF1 gene (3).

Fifth aspect present invention relates to the purposes of miR-24 inhibitor in the cancer of preparation treatment FAF1 gene unconventionality expression.

MiR-24 inhibitor refers in conjunction with miR-24 thus the material that it can be stoped to be combined with FAF1, to comprise its antisensenucleic acids, such as antisensenucleic acids shown in SEQ ID NO:4 in this article.Different modifications can be implemented to antisensenucleic acids, lock the 2 '-methoxyl group modification etc. occurred in nucleotide modification and other document as used in this article, or modify for sites different on antisensenucleic acids.Generally can modify base methoxy each on the antisense sequences of miR-24.Also can the some sites of selective modification.Specifically can list of references: Angie M.Cheng, Mike W.Byrom, JeffreyShelton and Lance P.Ford.; " Antisense inhibition of human miRNAs and indicationsfor an involvement of miRNA in cell growth and apoptosis "; Nucleic Acids Res., 2005; 33 (4): 1290 – 1297; And Wang Q, Huang Z, Xue H, Jin C, Ju XL, Han JD and ChenYG, " MicroRNA miR-24inhibits erythropoiesis by targeting activin type I receptorALK4 ", Blood, 2008Jan15; 111 (2): 588-95.Epub2007Sep28.

In one embodiment, the antisensenucleic acids of miR-24 is the antisensenucleic acids for miR-24 2-8 position (from 5 ' to 3 ') base, and its sequence is 5 '-CTGAGCC-3 '.The pairing base corresponding to miR-24 correspondence position can be added in any one or both sides of this sequence.Such as, described antisensenucleic acids can be the sequence sequence shown in SEQ ID NO:4 from 3 ' the 2nd to the 8th, 9,10,11,12,13,14,15,16,17,18,19,20,21 or 22 bit bases, or the sequence of SEQ ID NO:4 from 3 ' the 1st to the 8th, 9,10,11,12,13,14,15,16,17,18,19,20 or 21 bit bases.

The present invention includes SEQ ID NO:4 and derived the above-mentioned miR-24 antisensenucleic acids obtained by SEQ ID NO:4, and the expression cassette containing arbitrary above-mentioned antisensenucleic acids.

Sixth aspect present invention comprises a kind of pharmaceutical composition, the miR-24 inhibitor that said composition contains safe and effective amount and/or the FAF1 gene do not suddenlyd change or the expression vector containing this gene, FAF1 gene of the present invention or containing the carrier of this gene and/or the expression product (i.e. FAF1 albumen) of FAF1 gene and pharmaceutically acceptable carrier or excipient.As used herein, the composition of " pharmaceutically acceptable " is applicable to people and/or mammal and without excessive bad side reaction (as toxicity, stimulation and allergy), namely has the material of rational benefit/risk ratio.Term " pharmaceutically acceptable carrier " refers to the carrier being used for the treatment of agent administration, comprises various excipient and diluent.This kind of carrier comprises (but being not limited to): saline, buffer, glucose, water, glycerol, ethanol and combination thereof.Usual pharmaceutical preparation should match with administering mode, and pharmaceutical composition of the present invention can be made into injection form, such as, be prepared by conventional method with normal saline or the aqueous solution containing glucose and other adjuvant.Described pharmaceutical composition should aseptically manufacture.The dosage of active component is treatment effective dose.

Can also various suitable chemotherapeutics be contained in pharmaceutical composition of the present invention, such as, be used for the treatment of the chemotherapeutics of the cancer of various FAF1 gene unconventionality expression, the reagent star born of the same parents rhzomorph, 5-fluorouracil, mitomycin etc. of such as cell death inducing.

In a preferred embodiment, pharmaceutical composition of the present invention contains inhibitor and the cell death inducer of miR-24.In a detailed description of the invention, described derivant is star born of the same parents rhzomorphs.

In a preferred embodiment, pharmaceutical composition of the present invention contains the inhibitor of miR-24 and the overexpression vector of FAF1 gene.FAF1 gene in described overexpression vector can be the FAF1 gene do not suddenlyd change.

Seventh aspect present invention relates to FAF1 gene or its expression product is treated in preparation or prevents by the purposes in the medicine of the cancer of FAF1 gene unconventionality expression.Described cancer includes but not limited to malignant mesothe, hormone-independent prostate cancer, cervical cancer, gastric cancer, colon cancer and cancer of pancreas etc.In this respect, there is the gene of samesense mutation in the binding site that described FAF1 gene comprises miR-24 of the present invention, also comprises the gene of described binding site without sudden change.Especially, the present invention includes FAF1 gene or the purposes of its expression product in the medicine preparing treatment or prevention carcinoma of prostate.

Eighth aspect present invention provides a kind of method of the animal model for the preparation of research cancer mechanism and thus obtained animal model.The method comprises and regulating the FAF1 gene of this animal, thus makes this animal grow tumor, and carries out tumor research with this animal model, comprises drug screening etc.The present invention also comprises the medicine of the cancer using this animal model screening treatment FAF1 mediation, comprise and give this animal model medicine to be screened, and judge that can this medicine make tumor regression or the healing of this animal model, the medicine of the tumor regression of animal model or healing wherein can be made to be required medicine.Medicine both can be nucleic acid molecules, also can be chemical compound molecule.The method given both had comprised and directly having given, and such as oral, injection etc., also comprise and adopt giving of the method for the biotechnology such as genetic engineering.The method that those skilled in the art screen new drug to this employing animal model for tumour is known.

Ninth aspect present invention also relates to a kind of test kit, and this test kit contains miR-24 inhibitor and the reagent needed for this inhibitor of transfection.Optionally, this test kit also comprises the description using this miR-24 inhibitor transfecting animal cells.Or test kit contains mutagenic agent, for implementing samesense mutation to two miR-24 binding sites on the FAF1 gene ORF frame of cell, and described mutagenic agent how is used to implement the description of described sudden change.Or, this test kit comprises the inhibitor of miR-24 simultaneously, reagent needed for this inhibitor of transfection, and for implementing mutagenic agent or the mutation instrument of samesense mutationes to two miR-24 binding sites on the FAF1 gene ORF frame of cell, and the optional description about transfection and the description etc. about mutation.

Method of the present invention comprises the method for non-treatment object.Although it will be apparent to one skilled in the art that above-mentioned in certain in the content that describes may in other is other in do not describe, described content is equally also applicable to the technical scheme of described other side; Such as, the content describing transfection in second aspect present invention equally also can be used for other side, etc.That is, by above-mentioned each concrete in each specific features of describing combine the technical scheme of gained also within the scope of the invention.Hereafter the mode with specific embodiment is described the present invention.Following embodiment is only illustrative, and not restrictive.Those skilled in the art, when not departing from the application's spirit and scope, can adopt various substitute mode and combination thereof to implement the technical scheme of the application.

Embodiment 1: use the antisensenucleic acids of miR-24 (miR-24-ASO, SEQ ID NO:4) can apoptosis be caused and affect the propagation of DU-145 cell in DU-145 cell

In DU-145 cell, use the miR-24-ASO(of Interfer IN transfection reagent (PolyPlus-transfection) transfection 20nM concentration to be synthesized by TaKaRa company, sequence is 5 '-ctGTTCCTgctgAACTGAgcCA-3 ', i.e. SEQID NO:4, wherein lower case represent lock nucleic acid marking position), transfection used PI(Sigma after 48 hours) and marked FITC annexin V (Sigma) dye.By flow cytomery, find that miR-24-ASO can cause the apoptosis of DU-145 cell, the ratio of apoptosis is about about 10%, and (Fig. 1 a).

In order to detect the DU-145 cell of transfection miR-24-ASO further to the tolerance degree of apoptosis-inducing agent star born of the same parents rhzomorph, in DU-145 cell, use the miR-24-ASO of Interfer IN transfection reagent transfection 20nM concentration, transfection adds apoptosis-induced 2 hours of the star born of the same parents rhzomorph (Sigma) of middle concentration 1 μM for 48 hours in backward culture fluid.Found that the apoptosis ratio of transfection miR-24-ASO is about 49%, and do not have the apoptosis ratio of transfection to only have about 12%.Prove that the sensitivity of DU-145 cell to star born of the same parents rhzomorph of transfection miR-24-ASO significantly strengthens (Fig. 1 b).

Then, we have detected miR-24-ASO further on the apoptotic impact of DU-145.Result shows, and in transfection after miR-24-ASO, the multiplication capacity of DU-145 cell obviously weakens (Fig. 1 c).Contaminated miR-24-ASO and miR-24-mimics(Ambion at DU-145 transit cell) after have detected the ripe level of caspase8.Result shows transfection miR-24-ASO with the caspase8 showed increased of after ripening.The caspase8 level of cutting substance obviously reduces.Illustrate that miR-24-ASO may be by caspase8 path cell death inducing (Fig. 1 d).

Embodiment 2:miR-24 is by regulating and controlling FAF1 gene in conjunction with two target sites in the open reading frame (ORF) of FAF1 gene

Use following experimental technique, we have found the target gene that FAF1 gene may be miR-24.

Use the RNA22 software of IBM Corporation, the binding site of miR-24 is not predicted in the 3 ' untranslated region of FAF1, but having found the binding site of two miR-24 in the open reading frame of FAF1, is SEQ ID NO:1 1650-1670 position and 2128-2148 position respectively.Found by sequence analysis, the binding site of miR-24 is all very conservative in eight species such as people, Mus, cattle, especially 5 ' end the 2 to the 8 " seed " region combined of miRNA is nearly all (the seeing below) of guarding completely, further illustrates that miR-24 can (Fig. 2 a) by these two sites regulation and control FAF1 genes.

3'-GACAAGGACGACUUGACUCGGU-5' has-miR24

|||||||||||||||||

5'- uGUU cUuA u--- cUGAG cA-3' homo sapiens (Homo sapiens) B-1

G caAGC cCUGCcUC- cUGAGCCAhomo sapiens (Homo sapiens) B-2

U cUGa ua- uG cUC a uGAGCCAmouse (Mus musculus) B-1

ccA uUg uU ccU au cUGAG cAmouse (Mus musculus) B-2

G ca ggC c uGCcUC- cUGAGCCAcattle (Bos taurus) B-1

cUa uUg cU cUu au cUGAG cAcattle (Bos taurus) B-2

G caAGC cCUGCcUC- cUGAGCCAchimpanzee (Pan troglodytes)

cUa uUg cU cUu au cUGAG cAhorse (Equus caballus)

cUa uUg cU caU ac cUGAG cAafrica Pipa pipa (Xenopus tropicali)

cUa uUg cU caU ac cUGAG cAafrica xenopus (Xenopus laevis)

CC cc gcCG ca gCg g- cUGAGCCAfruit bat (Drosophila melano)

In above-mentioned sequence alignment, boldface type " U " represents that G:U matches, and underscore represents the sequence of complete complementary.

In order to verify this prediction further, the sequence clone containing this two basic change site has been arrived 3 ' untranslated region of the luciferase of pGL3 plasmid (Promega) by us.Then the plasmid transfection containing binding site entered 293T cell and after 24 hours, relative luciferase activity detected.Result display relative to transfection NC-mimics(Ambion) cell, in the cell of transfection miR-24-mimics, the relative activity of luciferase obviously reduces (Fig. 2 b).This illustrates that miR-24 can be incorporated into two basic change site that FAF1 gene is predicted and regulates and controls FAF1.

Whether regulate and control FAF1 gene in body to detect miR-24, we use pcDNA3.1-FAF1 plasmid overexpression FAF1 gene in 293T cell, and miR-24-mimics and NC-mimics of transfection simultaneously.In brief, use the method for PCR, with the cDNA library of people for template by FAF1 gene amplification rear clone to pCDNA3.1 plasmid, obtain pcDNA3.1-FAF1 plasmid, then use transfection reagent Lipofectamin2000 (Invitrogen) be transfected into cell.

This pcDNA3.1-FAF1 plasmid of following structure: between EcoRI and XhoI site FAF1cDNA (NM_007051) being cloned into pcDNA3.1 (-) (Invitrogen).Use following primer from human cDNA library, clone this FAF1cDNA:

5′-GAACTCGAGATTGGCGTCCAACATGGAC-3′ （SEQ ID NO:5）

5′-GCGCGAATTCTTACTCTTTTGCTTCAAGG-3′（SEQ ID NO:6）

Synthesis binding site is also cloned in 3 ' UTR region of the middle luciferase of pGL3 luciferase carrier (Promega).Then as mentioned before, by obtained construction and PRL-SV40 and miR-24-mimics or NC-mimics(Ambion) cotransfection enters in 293T cell.After 48 hours, double fluorescent enzyme reporter detection system (Promega) is used to measure Luciferase activity (meansigma methodss of three groups of parallel tests).

Result shows, and in transfection, in the cell of miR-24-mimics, the expression of FAF1 is starkly lower than cell and the only transfection cell of pcDNA3.1-FAF1 plasmid of transfection NC-mimics.And the suppression of miR-24 to FAF1 protein level is dose-dependent (Fig. 2 d).Simultaneously, we are transfection miR-24-ASO again after pcDNA3.1-FAF1 plasmid and miR-24-mimics in transfection, and synthesized by TaKaRa company with NC-ASO(, 5 '-caCTTATCagtcAGACCAtcGT-3 ', SEQ ID NO:11, lower case represents the site of LNA labelling) as reference, found that transfection miR-24-ASO has obviously recovered the downward (Fig. 2 c) of miR-24 to FAF1 gene.These results prove, miR-24 can regulate and control FAF1 gene in vivo.

In order to prove that miR-24 is regulated and controled FAF1 by the two basic change site of prediction further, we have especially done samesense mutation in " seed " region to two sites of prediction on FAF1: the method for being enclosed by plasmid amplification one by PCR, we construct some samesense mutationes at the miR-24 calmodulin binding domain CaM of FAF1 gene.Employ following sequence as PCR primer:

FAF1-Mu-B1-FW:5′-TTAGCTACCTCACGCAAAATTTTATAACCTGGGCTT-3′ （SEQ ID NO:7）

FAF1-Mu-B1-RV:5′-TGCGTGAGGTAGCTAACAATGGATTCAGCACAAAG-3′ （SEQ ID NO:8）

FAF1-Mu-B2-FW:5′-CTCCCTCCGGAACCTAAGGAAGAAAATGCTGAGCCTG-3′ （SEQ ID NO:9）

FAF1-Mu-B2-RV:5′-TTAGGTTCCGGAGGGAGGGCTTGCTCTAAGGACAG-3′ （SEQ ID NO:10）

Then the FAF1 gene (sudden change occurred is with FAF1-M1 and FAF1-M2 of embodiment 4) of sudden change is entered to 293T transit cell, proceed to the miR-24-mimics of variable concentrations simultaneously, result shows, the FAF1 gene suddenlyd change after turning 1nM or 5nMmiR-24-mimics is not lowered, and after proceeding to 20nM miR-24-mimics, FAF1 gene starts to lower.The ability of regulation and control of miR-24 to the FAF1 gene of sudden change obviously weakens (Fig. 2 e).These results illustrate that miR-24 can by the expression of the two basic change site regulation and control FAF1 gene of prediction.

Embodiment 3:miR-24 is by the apoptosis of targeting FAF1 gene regulation DU-145 cell

Whether undertaken by FAF1 gene the apoptotic regulation and control of DU-145 to detect miR-24, we distinguish the different reagent of cotransfection in DU-145 cell.Found that transfection pcDNA3.1-FAF1 overexpression FAF1 gene can to the apoptosis suppressing the expression of miR-24 equally to cause DU-145 cell.Then we transfection miR-24-ASO suppresses the expression of miR-24, simultaneously cotransfection pcDNA3.1-FAF1 overexpression FAF1.Found that: the apoptosis (after 48 hours about 70%) that inhibit overexpression FAF1 gene after miR-24 can induce DU-145 cell very efficiently.For verify further miR-24 can control FAF1 to the apoptotic regulation and control of DU-145; we have proceeded to miR-24-mimics overexpression miR-24 while overexpression FAF1, found that the DU-145 cell not apoptosis that overexpression miR-24 can well protect FAF1 to induce.These results prove that miR-24 carries out by the apoptosis of targeting FAF1 gene pairs DU-145 cell (Fig. 3) of regulating and controlling.

Embodiment 4: two miR-24 binding sites on samesense mutation FAF1 gene can be apoptosis-induced further

In order to confirm that miR-24 is undertaken by the two basic change site be attached on the ORF frame of FAF1 gene on the apoptotic impact of DU-145 further, by the overexpression vector pcDNA3.1-FAF1-M12 transfection DU-145 cell of sudden change.

The FAF1 overexpression vector pcDNA3.1-FAF1-M12 of two site seed region sudden changes that following structure miR-24 combines: use PCR method to build in two steps.The primer that sudden change uses is respectively:

FAF1-Mu-B1-FW:5′-TTAGCTACCTCACGCAAAATTTTATAACCTGGGCTT-3′ （SEQ ID NO:7）；

FAF1-Mu-B1-RV:5′-TGCGTGAGGTAGCTAACAATGGATTCAGCACAAAG-3′ （SEQ ID NO:8）；

FAF1-Mu-B2-FW:5′-CTCCCTCCGGAACCTAAGGAAGAAAATGCTGAGCCTG-3′（SEQ ID NO:9）；

FAF1-Mu-B2-RV:5′-TTAGGTTCCGGAGGGAGGGCTTGCTCTAAGGACAG-3′ （SEQ ID NO:10）。

With pcDNA3.1-FAF1 plasmid (building as described in Example 2) for template, build the FAF1 expression plasmid pcDNA3.1-FAF1-M1 of first binding site sudden change.PCR reaction system is:

10×KOD Buffer 5μl

dNTP（2mM） 5μl

MgSO ₄（20mM） 2μl

FAF1-Mu-B1-FW（20μM） 2μl

FAF1-Mu-B1-RV（20μM） 2μl

Template (pcDNA3.1-FAF1 plasmid) 1 μ l

KOD plus 1μl

Add H ₂o to cumulative volume 50 μ l

Eppendorf company PCR instrument two-step method is used to react.PCR reaction condition is:

1) 94 DEG C, 5 minutes; 2) 94 DEG C, 1 minute; 3) 68 DEG C, 10 minutes; 4) 2 are returned), carry out 20 and take turns; 5) 68 DEG C, 20 minutes; 7) 10 DEG C are maintained.

PCR rear electrophoresis glue reclaims.DpnI restricted enzyme 37 DEG C of enzyme action are used after 4 hours, to transform DH5 α bacterial strain.Qualification obtains the FAF1 expression vector pcDNA3.1-FAF1-M1 of first binding site sudden change.Then reaction system and reaction condition that structure first binding site is identical is used, with pcDNA3.1-FAF1-M1 plasmid for template, with FAF1-Mu-B2-FW and FAF1-Mu-B2-RV for primer builds the FAF1 expression vector pcDNA3.1-FAF1-M12 that there is sudden change in two sites.What occur sports:

5'-uuguuAGCuaC---cuCaCGca-3' FAF1-M1

5'-gcaagcccugccuccGgaAccU-3' FAF1-M2

FAF1-M1 and FAF1-M2 is the sudden change in corresponding homo sapiens B-1 and homo sapiens B-2 two basic change site respectively.Wherein capitalization represents the site of sudden change.454-2406 position in concrete sequence visible SEQ ID NO:12.

By the overexpression vector pcDNA3.1-FAF1-M12 transfection DU-145 cell of sudden change, the FAF1 of result display sudden change can induce the DU-145 apoptosis of about about 34%, the apoptosis that the do not suddenly change FAF1 mutually commensurability far above transfection induces after 36 hours.Then whether we demonstrate again miR-24 further and can regulate and control the FAF1 gene of sudden change.We are cotransfection miR-24-mimics while pcDNA3.1-FAF1-M12 in transfection; although found that in the experiment above, overexpression miR-24 well protects the DU-145 cell not apoptosis of overexpression FAF1, the DU-145 apoptosis that miR-24 overexpression but can not stop the FAF1 overexpression of sudden change to cause.These results show that miR-24 is (Fig. 4) that the apoptosis of two basic change site to DU-145 cell by being attached on the ORF frame of FAF1 gene regulates and controls.

Embodiment 5:miR-24-ASO can induce HeLa apoptosis equally and affect the propagation of HeLa cell

Then, we have contaminated the expression of miR-24-ASO suppression miR-24 at HeLa transit cell, have detected the impact of miR-24-ASO on HeLa cell proliferation simultaneously.The transfection of result display miR-24-ASO inhibits the propagation of HeLa cell.The star born of the same parents rhzomorph induction HeLa apoptosis later adding 1nM for 48 hours of we miR-24-ASO in transfection, after found that the expression suppressing miR-24, the toleration of HeLa cell to star born of the same parents rhzomorph obviously strengthen.These data tell us, and miR-24 may not only be confined to DU-145 cell to apoptotic regulation and control.The apoptosis (Fig. 5) of kinds of tumor cells may be caused after suppressing the expression of miR-24.

Because DU-145 cell is the insensitive cell of hormone, therefore these data provide a possible target spot to we providing carcinoma of prostate even a treatment for some other tumor for the insensitive class of hormone.

Embodiment 6:miR-24-ASO suppresses the expression of the endogenous miR-24 of stomach cancer cell

Use Interfer IN transfection reagent to suppress the expression of endogenous miR-24 respectively to transfection 20nMmiR-24-ASO in two strain stomach cancer cell HGC-27 and MGC-803, the miR-24-ASO that transfection simultaneously " seed site " suddenlys change, namely NC-ASO in contrast.Basis of microscopic observation cell after transfection 36 hours, after found that the expression suppressing endogenous miR-24, all there is obvious apoptosis relative to matched group in this two strains stomach cancer cell.Use Annexin V-FITC and PI to dye, then use flow cytometer to carry out quantitative analysis to apoptosis degree.Found that, the apoptosis that two strain stomach cancer cell HGC-27 and MGC-803 transfection miR-24-ASO occur all obviously increases relative to the level of apoptosis of matched group.The early apoptosis rate of the HGC-27 after transfection miR-24-ASO is about 14%, and late apoptic rate is about 10%.About high relative to matched group about 3 times.The early apoptosis rate of the MGC-803 after transfection miR-24-ASO is about 13%, and late apoptic rate is about 11%.About high respectively relative to matched group 2 times and about 5 times.Result display in figs. 6 and 7.

Embodiment 7:miR-24-ASO and AOS-miR-24 treats the animal model experiment of tumor

1. materials and methods

(1) sequence of miR-24 is as shown in SEQ ID NO:3, is a RNA sequence only having 22 nucleotide.MiR-24-ASO is the complementation of the antisensenucleic acids of miR-24, its sequence and miR-24, as shown in SEQ ID NO:4.The sequence of miR-24-ASO is fixing.But can select modify different editings or select different modification modes in base, except modifying except lock nucleic acid used in literary composition, it is also conventional modification that 2 ' methoxy is modified.

(2) cell culture and transfection

293T, HeLa and DU-145 cell is obtained, at 37 DEG C, 5%CO in DMEM or DMEM/F12 supplementing 10% hyclone (FBS) from the cell bank of the Chinese Academy of Sciences ₂lower these cells of cultivation.

For transient transfection, with Lipofectamine2000(Invitrogene) by degree of being paved be 70% cell transfecting on the synthesis miRNA-mimics of variable concentrations, negative control (NC-mimics) (Ambion), the widow-ribonucleotide miR-24-ASO of synthesis LNA labelling and the NC-ASO(TaKaRa of LNA labelling) and the plasmid that is worth according to standard scheme.After transfection, at different time points collecting cell, carry out biochemistry or biological analysis.

The sequence of the NC-ASO of LNA labelling is 5 '-caCTTATCagtcAGACCAtcGT-3 ' (SEQ ID NO:11); The sequence of the oligoribonucleotide miR-24-ASO of LNA labelling is 5 '-ctGTTCCTgctgAACTGAgcCA-3 '; In above-mentioned sequence, lowercase alphabet shows the site of LNA labelling.

(3) plasmid construction

5′-GAACTCGAGATTGGCGTCCAACATGGAC-3′ （SEQ ID NO:5）

5′-GCGCGAATTCTTACTCTTTTGCTTCAAGG-3′（SEQ ID NO:6）。

Adopt aforementioned primer SEQ ID NOS:7-10, be structured in miR-24 binding site the several FAF1 mutants that there is sudden change by PCR method.

(4) luciferase detects

(5) apoptosis analysis

With different synthetic oligonucleotides or different carrier transfection DU-145 cells and HeLa cell.Extra cultivation is after 36 hours or 48 hours, collecting cell, with the anti-annexin-V antibody staining of iodate third ingot and FITC labelling, then uses facs analysis.

(6) cell proliferation detects

Use Cell counting Kit-8(Dojindo) carry out cell proliferation detection.Cell is seeded on 24 hole flat boards, every hole about 5 × 10 ⁴individual cell, and cultivate in growth medium.At the appointed time put WST-8 (2-(2-methoxy-nitro the phenyl)-3-(4-nitrobenzophenone reduced)-5-(2,4-bis-sulfophenyl)-2H-tetrazolium monosodium salt) and absorbance (450nm) measure cell quantity in three groups of parallel test holes.

(7) Protein Extraction and Western blotting

With 1 × SDS Loading buffer (Sigma) collecting cell.Protein isolate in SDS-polyacrylamide gel, then transfers to pvdf membrane.Then, with TTBS(containing the Tris buffer of 1% tween) at room temperature to blockade film 1 hour with 5% skim milk powder (using PBST to dissolve), then make it at the TTBS(containing first antibody containing the Tris buffer of 1% tween) in 1% defatted milk powder in 4 DEG C spend the night.First antibody is detected with the second antibody (Sigma) of peroxidase conjugate and chemiluminescence (ECL) (Pierce).Use following first antibody: the anti-FAF(Cell signaling of rabbit) and GAPDH(Abcam).

(8) animal model test

First inoculate prostate cancer tumor block than tumor cell suspension at nude mice by subcutaneous, set up tumor model of prostate cancer.

Become the viral vector of injecting miR-24-ASO or FAF1-M12 gene after tumor in the tumor block of experimental mice or both inject simultaneously, simultaneously with NC-ASO and/or not containing FAF1 gene viral vector as a control group.

Behind some skies, take out tumor mass and observe Tumor size and shape.The carcinoma of prostate bone marrow transfer case of test experience group and nude mice of control group is to verify the effect of medicine to carcinoma of prostate transfer ability in addition.

2. experimental result

Expect that tumor block in the Mice Body after the viral vector of injection miR-24-ASO or FAF1-M12 gene or both are injected the simultaneously tumor block relative to matched group is at volume with can diminish qualitatively.The neoplasm metastasis ability of injectable drug group obviously reduces relative to matched group.

Discuss

The application finds that miR-24 by being attached to the expression of the CDS regional control FAF1 of FAF1, thus can regulate and control the apoptosis of DU-145 cell further.Find that miR-24-ASO can induce the apoptosis of DU-145 cell and HeLa cell simultaneously, deepened our understanding to Faf1 regulating cell apoptosis pathway.

DU-145 cell is the prostate cancer cell line of the people of the classics separated from carcinoma of prostate brain metastes.It has the transitivity of middle energy, does not express prostate specific antigen (PSA) and is the prostate gland cancer cell of non-hormone dependence.For hormone-independent prostate cancer, good therapeutic effect can be reached by hormonotherapies such as castration, androgen antagonist medicines.But to non-hormone dependence carcinoma of prostate, still there is no reasonable therapy at present.The apoptosis that the application finds miR-24-ASO, pcDNA3.1-FAF1-M12 and the FAF1 gene of overexpression all can well induce DU-145 cell after miR-24-ASO suppresses miR-24 expression.Especially, both after, apoptosis rate reaches 34% and about 70% respectively.These experimental results provide good clue for we treat non-hormone dependence carcinoma of prostate by miR-24/FAF1 path.

MiR24 is a microRNA very large with human tumor dependency.Be not only because he can suppress the expression of tumor suppressor gene p16, some researchs simultaneously also find the unconventionality expression of miR-24 in some tumor types.These all show miR-24 may be an oncogene microRNA.Our result of study finds that miR-24 can regulate and control the apoptosis of DU-145 cell and HeLa cell, illustrates that miR-24/FAF1 path modulate tumor apoptosis may be a general apoptosis of tumor cells regulatory mechanism.

In sum, our research finds that miR-24 can be regulated and controled FAF1 gene by two basic change site on the ORF frame that is attached to FAF1.MiR-24-ASO suppresses the expression of miR-24 can induce the apoptosis of DU-145 cell simultaneously.Our research further demonstrates microRNA and can be incorporated into regional control beyond 3 ' untranslated region the theory of memory.Meanwhile, our research is that the treatment of non-hormone dependence carcinoma of prostate and other cancer is carried and controlled potential target site and medicinal application prospect.

Claims

The purposes of 1.miR-24 in the compositions preparing the carcinoma of prostate for the treatment of or preventing FAF1 gene unconventionality expression or test kit.
2. purposes as claimed in claim 1, it is characterized in that, described carcinoma of prostate is hormone-independent prostate cancer.
The purposes of 3.miR-24 inhibitor in the compositions preparing the carcinoma of prostate for the treatment of or preventing FAF1 gene unconventionality expression or test kit.
4. purposes as claimed in claim 3, it is characterized in that, described carcinoma of prostate is hormone-independent prostate cancer.
5. the purposes as described in claim 3 or 4, is characterized in that, described miR-24 inhibitor is the antisensenucleic acids of miR-24.
6. purposes as claimed in claim 5, it is characterized in that, described antisensenucleic acids is as shown in SEQ ID NO:4.
The antisensenucleic acids of 7.miR-24, is characterized in that, described anti sense nucleotide sequence is 5 '-ctGTTCCTgctgAACTGAgcCA-3 ', and wherein lower case represents the position of lock nucleic acid marking.